Effects of different iron treatments on wine grape berry quality and peel flavonoid contents

Abstract In this study, eight‐year‐old wine grape plants (Cabernet Sauvignon) were subjected to five different iron treatments: ferrous sulfate, ferric ethylenediaminetetraacetic acid (EDTA‐Fe), ferric citrate, ferric gluconate, and ferric sugar alcohol, and conventional fertilization. Foliar spraying with clear water was used as the control treatment. The effects of different iron treatments on berry quality and flavonoid accumulation in grape peels were explored. All five iron treatments affected the sugar, acid, and peel flavonoid contents of grape berries, but the contents varied greatly among the different iron treatments. Foliar spraying with iron increased berry sugar content and reduced acid content. In addition, foliar spraying with ferrous sulfate, EDTA‐Fe, ferric gluconate, and ferric sugar alcohol reduced the total anthocyanin, flavanol, and flavonol contents in the peel. The unique flavonoid monomer content of the peel was significantly higher under ferric citrate treatment than under the control and other iron treatments. Moreover, the results showed that foliar spraying with ferric citrate balanced the berry sugar–acid ratio and also increased the anthocyanin, flavanol, and flavonol contents of the grape peel, thereby improving the overall nutritional status of the berries and the final wine quality. The results obtained in this study demonstrate that different iron treatments could improve grape berry quality and clarify the effects of different exogenous iron treatments.


| INTRODUC TI ON
The eastern foothills of Helan Mountain in Ningxia are among the best ecological regions for wine grapes in China. However, the fertilization method used for wine grape cultivation is inclined to supplement traditional macronutrient fertilizers, ignoring the importance of trace elements. The mineral nutrient availability in the alkaline soil in this area is low, the effective iron content is far lower than the national average, and the mobility of iron in plants is not strong, making it difficult for various elements that were originally insufficient to meet the nutritional requirements for the normal growth of grapes.
Foliar fertilization is one of the main methods used to improve the berry yield and quality during grape cultivation. Foliar application is the most rapid and effective method for satisfying the specific nutritional needs of plants (Fernandez et al., 2009). The effect of foliar iron application on the composition of grapes is highly dependent on the grape variety and the duration and form of iron used (Abadia et al., 2002;Alvarez-Fernàndez et al., 2006;Yunta et al., 2013). Iron fertilizers can be divided into three categories: inorganic, organic compound, and chelated iron fertilizers. Inorganic iron fertilizer is inexpensive and has a fast fertilizer effect, but it is easily oxidized, resulting in an unsustainable fertilizer effect. Ferrous sulfate is the most common fertilizer. Organic compound iron fertilizers are relatively stable and easily degradable, and the most common ones are ferric gluconate and ferric sugar alcohol. The chelated iron fertilizers are mainly ferric ethylenediaminetetraacetic acid (EDTA-Fe), ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA-Fe), and ferric citrate. Furthermore, studies have shown that the application of chelated iron can significantly increase the soluble sugar and phenolic acid contents of grapes under alkaline soil conditions (Karimi et al., 2019).
The quality of grapes is related to the balance between primary and secondary metabolites. Glucose and fructose are the main sugars in berries, and the contents of these primary metabolites are affected by variety, climate, and nutritional status, while the accumulation of sugar in berries can improve the volatile aromatic compound content (Ali et al., 2010;Zheng et al., 2009). Phenols are important components that determine the quality of grape berries and have various functions in plants, including as antioxidants, protecting against ultraviolet (UV) radiation, and combating pathogenic infections (Tian et al., 2019). Studies have shown that iron is related to the function of phenolic synthase; thus, iron deficiency affects the synthesis of phenolic compounds via the shikimate pathway (Bavaresco et al., 2005). In addition, iron concentration can affect pectin degradation and antioxidant capacity of phenolic compounds (Vidot et al., 2020). Many phenolic compounds are found in grapes at high concentrations. These phenolic compounds can be divided into flavonoids and nonflavonoids, where flavonoids comprise flavanols, flavonols, and anthocyanins, and nonflavonoids include resveratrol, benzoic acid, and cinnamic acid (Liang et al., 2013). The expression levels of genes related to anthocyanin biosynthesis are affected by developmental and environmental factors, including temperature, light, sugar content, and mineral content. Karimi et al. (2019) found that exogenous iron could regulate the anthocyanin content of berries. Zheng et al. (2009) showed that carbohydrates can promote the expression of flavanone 3-hydroxylase (EC 1.14.11.9), which is a key enzyme in anthocyanin synthesis, thereby increasing berry anthocyanin content.
Flavonols are produced by the flavonoid biosynthesis pathway and mainly comprise quercetin, myricetin, kaempferol, and isorhamnetin (Mattivi et al., 2006). Flavan-3-alcohol is a basic component of proanthocyanidins and condensed tannins, including catechin, epicatechin, gallocatechin, epigallocatechin, and epigallocatechin gallate. Flavonoids and flavanols are mainly distributed in the grape seeds, peel, and berry stems. Flavanols have important effects on astringency, bitterness, and structure of wine (Zimman et al., 2002). Foliar spraying with iron can increase the anthocyanin (Shi et al., 2017), flavonol, and flavanol (Shi et al., 2018) contents of grape berries. However, previous studies have not comprehensively investigated the effects of different forms of iron on the flavonoid content of grapes.
In this study, we investigated the effects of foliar treatment with different forms of iron on berry quality and flavonoid accumulation in the peel of Cabernet Sauvignon grapes, thereby providing a theoretical basis for improving wine grape cultivation in alkaline soil at the eastern foothills of the Helan Mountain in China.

| Test materials and experimental design
The experiment was conducted from April to October, 2021 at Lilan Winery (105°58′20′′E, 38°16′38′′N), which is in a wine grapeproducing area at the eastern foothills of the Helan Mountain in China. The study site was located at an altitude of 1129 m, with an annual average precipitation of 190 mm and a frost-free period of 180 days. Eight-year-old Cabernet Sauvignon grape vines were used in this study. The vine shape was "厂" and the row spacing was 0.6 × 3.5 m. The basal fertilizer, comprising organic sheep manure fertilizer, was ditched in early May and applied once at 10,500 kg hm −2 by drip irrigation. The soil type was calcareous gravel. Table 1 lists the chemical characteristics of the soil before the start of the experiment.
The experiment had a randomized block design with six treatments and three replicates for each treatment, with a total of 18 cells, and each cell area was 10.5 m 2 . Each iron fertilizer treatment was sprayed three times (on July 12, July 27, and August 11) with an electric sprayer at the grape expansion and color-changing stages, where a 5 L solution of each treatment was applied. Table 2 shows the total amounts of iron received for each treatment after the three applications. Excluding the different forms of iron fertilizer, all other treatments and management measures were consistent with those used in the vineyard. During the optimal grape harvest period, 15 representative grapes were randomly selected from each treatment and transported rapidly to the laboratory. One hundred grapes were immediately frozen in liquid nitrogen and ground to determine berry quality. For each treatment, 30 grapes were randomly selected, washed with distilled water, peeled, frozen in liquid nitrogen, and stored at −80°C to determine the peel metabolites.

| Determination of grape berry quality
Frozen grape berries were ground to determine the total soluble solids (TSS), reducing sugar, and titratable acid contents (TAC). The TSS TA B L E 1 Chemical characteristics of the soil before the trial commenced (g kg −1 )

| Extraction of anthocyanin and flavonoid compounds from peel
Grape peel samples were vacuum dried and frozen for 24 h in a lyophilizer (Scientz-100F) and then ground (30 Hz, 1.5 min) into powder using a grinder (MM 400, Retsch

| Qualitative and quantitative analyses of anthocyanin and flavonoid compounds
The ion current intensities and retention times were compared with those of our self-developed "metware" database (MWDB database). Qualitative and quantitative analyses of the compounds were conducted according to the secondary spectrum information. The contents of different metabolites were analyzed according to the metabolite detection multimodal diagram.

| Statistical analysis
Microsoft Excel 2010 and SPSS 21.0 software were used to process and analyze the data. Origin2018 was used to plot the data.
Significant differences were accepted at p < .05 (n = 5). All data are expressed as the mean ± standard error.

Indexes Form of iron fertilizer Solution concentration (%) Iron fertilizer application rate (kg hm −2 ) Iron dosage (kg hm −2 )
Control ---  iron treatments also significantly increased the 100-berry weight compared with the control, but the differences in the weights were not significant between the different iron treatments.
The contents of the anthocyanin monomers such as Cyacte, Dpacet, Cycoum, Mv, and Decoum were the highest in the peel. The iron treatments significantly increased the content of Mv and its various morphological derivatives, but did not have significant effects on the contents of the anthocyanins such as Pn, Pndigl, and

| Effects of different iron treatments on flavanols and flavonols in grape peel
The flavonoids detected in grape berry skin are shown in Table 5, where 19 flavanols and 42 flavonols were detected. The abbreviations for these compounds are listed in Abbreviations: RS, reducing sugar (expressed as gram equivalent glucose L −1 ); TAC, titratable acid content (expressed as gram equivalent tartaric acid L −1 ); TSS, total soluble solids content (%); WB, weight of 100 berries (g).

| Principal component analysis of grape berry flavonoids
The different iron treatments had significant effects on the content and proportion of sugar acids and flavonoids in grape berries.
Principal component analysis (PCA) was conducted to determine the overall differences in flavonoids under different iron treatments.

| DISCUSS ION
Alvarez-Fernàndez et al., (2006) showed that iron deficiency decreases berry sugar content. The sugar-acid ratio is generally a measure of berry ripeness. In the present study, spraying with different forms of iron improved the quality of wine grapes, where TSS increased, and sugar content and TAC decreased, thereby increasing the sugar-acid ratio. The increase in the sugar-acid ratio in grape berries under the iron treatments indicates that iron can promote berry ripening, as also observed in pears ("Deveci" and "Santa Maria") (Ozturk et al., 2019), table grapes (cv. "Thompson Seedless") (Taghavi et al., 2020), and wine grapes (Vitis vinifera cv.) (Shi et al., 2017). Reductions in RuBisCO (ribulose-1,5-bisphosphate carboxylase-oxygenase) activity levels and lower chlorophyll and carotenoid contents in iron-deficient plants lead to a lower leaf CO 2 exchange rate and photosynthetic efficiency, which may explain the higher sugar content under iron treatment in the present study (Chen et al., 2004). We found that the glycolic acid contents of grape berries differed significantly among the iron treatments, while the TSS contents were also higher and the sugar contents were lower under FB2, FB3, FB4, and FB5 than under FB1. Organic chelated iron is a small molecule that is absorbed by the leaves when chelated by sugar alcohols and amino acids, thereby avoiding the oxidation and precipitation of ferrous sulfate when sprayed alone (FB1), facilitating the absorption of nutrient iron (Fernández & Ebert, 2005). In addition, sugar alcohols and amino acids are small organic molecules with good moisture retention, permeability, and ductility characteristics; thus, they can reduce the surface tension and improve the capacity of leaf surfaces to absorb iron (Singh et al., 2013). Tartaric acid and malic acid are the main organic acids present in grape berries.
We found that iron treatment decreased TAC, except for FB3 (ferric citrate), possibly because external application of iron promoted the accumulation of sugars and accelerated berry ripening, whereas the malic acid content of berries gradually decreased as the berry matured (Karimi et al., 2019). Malic and citric acids are the main substrates for plant respiration. The TSS and reducing sugar contents of berries increased under FB3 (ferric citrate), whereas TAC did not change compared with the control, probably because the externally applied ferric citrate was consumed by respiration to decrease the decomposition of malic acid; thus, TAC was higher under FB3 (ferric citrate) compared with other iron treatments (Chen et al., 2004;Schlegel et al., 2006). The weight of berries is determined by their size and density, which are important factors that affect the quality of grapes. The weights of the grape berries were significantly higher under the iron treatments than under the control, possibly because iron increased the metabolic activity of the plants. Iron deficiency during grape growth is known to reduce membrane integrity, leaf CO 2 content, exchange rate, and chlorophyll photosynthetic efficiency to inhibit the accumulation of dry matter, which may explain why iron treatment significantly increased the weight of the berries in the present study (Bertamini & Nedunchezhian, 2005).
The types and quantities of anthocyanins detected in grape peel in the present study were generally consistent with those previously reported (Arozarena et al., 2002;Mattivi et al., 2006;Shi et al., 2017).
Anthocyanins are important pigments in red grape. The proportions and quantities of anthocyanins were determined based on the specific variety and cultivation conditions. Studies have shown that iron is an important factor affecting anthocyanin synthesis (Ahmed et al., 1997). In the present study, we found that spraying different forms of iron increased the content of specific anthocyanins. In particular, foliar application of ferric citrate (FB3) and ferric gluconate Note: Different lowercase letters indicate significant differences between treatments, according to Tukey's HSD (honest significant difference) test (p < .05).

Flavonoids
Abbreviations   (FB4) significantly increased the contents of some individual anthocyanins and the total anthocyanins in the grape peel, probably because glucose, fructose, and sucrose can induce the accumulation of anthocyanin in grape berries (Zheng et al., 2009). Indeed, the sugar content of grape berries increased significantly under these two treatments, thereby promoting anthocyanin synthesis in grape peel.

TA B L E 5 Effects of different iron treatments on the flavanol and flavonol contents of grape peel
However, we also found that under treatment with ferrous sulfate (FB1), EDTA-Fe (FB2), and ferric sugar alcohol (FB5), the levels of some individual anthocyanins (Cyacet, Dpacet, and Cycoum) were lower than those in the control, resulting in the total anthocyanin content being lower than that in the control as well. In contrast, previous studies found that anthocyanin content increased under iron treatment (Ahmed et al., 1997;Singh et al., 2013)

| CONCLUSION
In this study, we found that five different foliar iron treatments affected the fructose, acid, and flavonoid contents of Cabernet Sauvignon grapes, and that the various iron treatments also had different effects. Spraying iron on the leaves could increase the sugar content and reduce the acid content of berries. However, spraying ferrous sulfate, EDTA-Fe, ferric gluconate, and ferric sugar alcohol on the leaves reduced the total anthocyanin, flavanol, and flavonol contents in the peel. In addition, the contents of specific flavonoid monomers were significantly higher in the grape peel under some iron treatments than in the control, as well as with the other iron treatments. However, our comprehensive study showed that foliar spraying with ferric citrate balanced the sugar-acid ratio in the berry and increased the anthocyanin, flavanol, and flavonol contents of the grape peel to further improve the quality of the grapes, thereby possibly enhancing the overall nutritional content of the berries and the final wine quality.

ACK N OWLED G M ENTS
This study was supported by the National Key Research and Development Project (2019YFD1002500) and Ningxia Natural Science Foundation (2020AAC03281). We thank our colleagues for their comments on this paper, and the journal's editors and anonymous reviewers for their critical reviews and comments regarding this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.